Most eukaryotic messenger RNA precursors (pre-mRNAs) must undergo co-transcriptional processing in the nucleus before they can function as mRNAs in the cytoplasm. The processing events include 5'capping, splicing, and 3'polyadenylation. The 3'polyadenylation of pre-mRNAs occurs in two steps - endonucleolytic cleavage of the pre-mRNA at a specific site near its 3'-end and then the addition of the poly(A) tail. While the cleavage reaction may appear to be simple biochemically, it requires a large number of protein factors for its execution, including the cleavage and polyadenylation specificity factor (CPSF) complex, the cleavage stimulation factor (CstF) complex, cleavage factors I and II, and poly(A) polymerase (PAP). The CPSF complex contains four subunits, CPSF-30, CPSF-73, CPSF-100, and CPSF-160, and the CstF complex contains three subunits, CstF-50, CstF-64, and CstF-77. Despite the characterization of this large number of proteins that are required for the cleavage reaction, the identity of the endonuclease that actually catalyzes the hydrolysis is currently unknown. Recent evidence suggests CPSF-73 could be the endonuclease for the cleavage reaction, although no direct experimental evidence demonstrating this activity is available. Moreover, there is currently little structural information on these important proteins. Only the structures of PAP and the RNA binding domain of CstF-64 are known. To fill this gap in our knowledge, we have recently determined the crystal structures of human CPSF-73, yeast CPSF-100, murine CstF-77, and carried out preliminary biochemical and mutagenesis studies to assess the information from the structures. These initial results set the stage for further biochemical, biophysical, and structural studies on these proteins with crucial roles in pre-mRNA 3'-end processing.